Methods for high throughput genotyping

ABSTRACT

Methods for genotyping polymorphisms using allele specific probes are disclosed. A training set is used to generate a model for each polymorphism to be interrogated. The training set is used to obtain an estimate of the asymmetry between an intensity measurement for a first allele and an intensity measurement for a second allele of the same polymorphism. The intensity measurement obtained for a test sample is adjusted using the estimate of asymmetry prior to using the intensity measurements to make a genotyping call. In preferred embodiments the adjustment is applied to polymorphisms that have a likelihood of being heterozygous that is above a specified threshold.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.12/638,939 filed Dec. 15, 2009, which is a continuation of U.S. patentapplication Ser. No. 11/608,233 filed Dec. 6, 2006, now U.S. Pat. No.7,634,363, and claims priority to U.S. Provisional Patent ApplicationNo. 60/748,427 filed Dec. 7, 2005, the entire disclosures of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention is related to methods of genotyping polymorphisms. Thepresent invention relates to computer systems, methods, and products forthe analysis of microarray hybridization data.

BACKGROUND OF THE INVENTION

The genetic sequences of different individuals are identical over themajority of the genome and vary on average only at about one base inevery 1000. The most common form of variation is a difference at asingle base, known as a single nucleotide polymorphism or SNP. At theseSNP positions some portion of the population will have one base whileanother portion of the population with have a different base. SNPs actas markers to locate regions of a genome that may be associated with aparticular phenotype, such as a risk for disease.

SUMMARY OF THE INVENTION

Methods for calling the genotype of a sample at a selected polymorphismin a sample using a genotyping array are disclosed. In preferred aspectsthe methods include a normalization step where an intensity measurementfor a first allele is adjusted to account for an asymmetry in intensitymeasurements between the first allele and a second allele that areobserved in a training set of samples.

In one aspect the steps include: obtaining intensity measurements forallele A and for allele B for a plurality of polymorphisms in aplurality of training samples, wherein the genotype of each polymorphismin the plurality in each training sample is known of known genotype,wherein the intensity measurements represent intensity of signalassociated with one or more features on said genotyping array, making agenotype call for each of a said polymorphisms in each of the trainingsamples using the intensity measurements for allele A and for allele Bobtained above, comparing the genotype call with the known genotype toidentify individuals where the correct genotype call was made, using theintensity measurements from the individuals identified above tocalculate a ratio of intensity measurement for allele A to intensitymeasurement for allele B, for the training samples for each sub-group ofAA, AB and BB to obtain an AA reference ratio, an AB reference ratio anda BB reference ratio for each of said polymorphisms; hybridizing a testsample to the genotyping array to obtain hybridization intensity valuesfor the A allele and for the B allele for each of said polymorphisms inthe test sample; calculating a ratio of the intensity measurement forthe A allele to the B allele for each of said polymorphisms in the testsample and compare the ratio to the reference ratios for AA, AB and BBobtained for that polymorphism above to determine the likelihood thatthe polymorphism is AB, identifying a subset of the polymorphisms in thetest sample that are likely to be AB, wherein a polymorphism isidentified as being likely to be AB if the likelihood that thepolymorphism is AB is greater than a selected threshold, adjusting theintensity measurement of the B allele by the reference ratio for the ABgroup for that polymorphism from the training set to obtain an adjustedintensity measurement for the B allele, for each polymorphism in thesubset of polymorphisms identified above; and generating a genotype callfor each polymorphisms identified above using the use the adjustedintensity measurement for the B allele.

In one aspect, the allele specific intensity measurements may bemeasurements of the amount of target hybridized to an allele specificprobe or set of probes. In another aspect, the allele specific intensitymeasurements may be measurements of the amount of signal incorporatedinto a probe in a template dependent primer extension assay such as asingle base extension assay or an allele specific primer extensionassay.

In another aspect computer software and systems to implement thedisclosed methods are contemplated.

The above implementations are not necessarily inclusive or exclusive ofeach other and may be combined in any manner that is non-conflicting andotherwise possible, whether they are presented in association with asame, or a different, aspect of implementation. The description of oneimplementation is not intended to be limiting with respect to otherimplementations. Also, any one or more function, step, operation, ortechnique described elsewhere in this specification may, in alternativeimplementations, be combined with any one or more function, step,operation, or technique described in the summary. Thus, the aboveimplementations are illustrative rather than limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and form a part ofthis specification, illustrate embodiments of the invention and,together with the description, serve to explain the principles of theinvention:

FIG. 1 is a flowchart for making heterozygous calls using intensitymeasurements from a training sample to adjust the intensity measurementfor one allele.

FIG. 2 is a flowchart of an embodiment using hybridization intensitiesfrom allele specific probes.

DETAILED DESCRIPTION OF THE INVENTION (A) General

The present invention has many preferred embodiments and relies on manypatents, applications and other references for details known to those ofthe art. Therefore, when a patent, application, or other reference iscited or repeated below, it should be understood that it is incorporatedby reference in its entirety for all purposes as well as for theproposition that is recited.

As used in this application, the singular form “a,” “an,” and “the”include plural references unless the context clearly dictates otherwise.For example, the term “an agent” includes a plurality of agents,including mixtures thereof.

An individual is not limited to a human being but may also be otherorganisms including but not limited to mammals, plants, bacteria, orcells derived from any of the above.

Throughout this disclosure, various aspects of this invention can bepresented in a range format. It should be understood that thedescription in range format is merely for convenience and brevity andshould not be construed as an inflexible limitation on the scope of theinvention. Accordingly, the description of a range should be consideredto have specifically disclosed all the possible subranges as well asindividual numerical values within that range. For example, descriptionof a range such as from 1 to 6 should be considered to have specificallydisclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numberswithin that range, for example, 1, 2, 3, 4, 5, and 6. This appliesregardless of the breadth of the range. All references to the functionlog default to e as the base (natural log) unless stated otherwise (suchas log₁₀).

The practice of the present invention may employ, unless otherwiseindicated, conventional techniques and descriptions of organicchemistry, polymer technology, molecular biology (including recombinanttechniques), cell biology, biochemistry, and immunology, which arewithin the skill of the art. Such conventional techniques includepolymer array synthesis, hybridization, ligation, and detection ofhybridization using a label. Specific illustrations of suitabletechniques can be had by reference to the example herein below. However,other equivalent conventional procedures can, of course, also be used.Such conventional techniques and descriptions can be found in standardlaboratory manuals such as Genome Analysis: A Laboratory Manual Series(Vols. I-IV), Using Antibodies: A Laboratory Manual, Cells: A LaboratoryManual, PCR Primer: A Laboratory Manual, and Molecular Cloning: ALaboratory Manual (all from Cold Spring Harbor Laboratory Press),Stryer, L. (1995) Biochemistry (4th Ed.) Freeman, New York, Gait,“Oligonucleotide Synthesis: A Practical Approach” 1984, IRL Press,London, Nelson and Cox (2000), Lehninger, Principles of Biochemistry3^(rd) Ed., W.H. Freeman Pub., New York, N.Y. and Berg et al. (2002)Biochemistry, 5^(th) Ed., W.H. Freeman Pub., New York, N.Y., all ofwhich are herein incorporated in their entirety by reference for allpurposes.

The present invention can employ solid substrates, including arrays insome preferred embodiments. Methods and techniques applicable to polymer(including protein) array synthesis have been described in U.S. Ser. No.09/536,841, WO 00/58516, U.S. Pat. Nos. 5,143,854, 5,242,974, 5,252,743,5,324,633, 5,384,261, 5,405,783, 5,424,186, 5,451,683, 5,482,867,5,491,074, 5,527,681, 5,550,215, 5,571,639, 5,578,832, 5,593,839,5,599,695, 5,624,711, 5,631,734, 5,795,716, 5,831,070, 5,837,832,5,856,101, 5,858,659, 5,936,324, 5,968,740, 5,974,164, 5,981,185,5,981,956, 6,025,601, 6,033,860, 6,040,193, 6,090,555, 6,136,269,6,269,846 and 6,428,752, in PCT Applications Nos. PCT/US99/00730(International Publication Number WO 99/36760) and PCT/US01/04285, whichare all incorporated herein by reference in their entirety for allpurposes.

Patents that describe synthesis techniques in specific embodimentsinclude U.S. Pat. Nos. 5,412,087, 6,147,205, 6,262,216, 6,310,189,5,889,165, and 5,959,098. Nucleic acid arrays are described in many ofthe above patents, but the same techniques are applied to polypeptidearrays.

Nucleic acid arrays that are useful in the present invention includethose that are commercially available from Affymetrix (Santa Clara,Calif.) under the brand name GeneChip®. Example arrays are shown on thewebsite at affymetrix.com.

The present invention also contemplates many uses for polymers attachedto solid substrates. These uses include gene expression monitoring,profiling, library screening, genotyping and diagnostics. Geneexpression monitoring, and profiling methods can be shown in U.S. Pat.Nos. 5,800,992, 6,013,449, 6,020,135, 6,033,860, 6,040,138, 6,177,248and 6,309,822. Genotyping and uses therefore are shown in U.S. Ser. No.60/319,253, 10/013,598, and U.S. Pat. Nos. 5,856,092, 6,300,063,5,858,659, 6,284,460, 6,361,947, 6,368,799 and 6,333,179. Other uses areembodied in U.S. Pat. Nos. 5,871,928, 5,902,723, 6,045,996, 5,541,061,and 6,197,506.

The present invention also contemplates sample preparation methods incertain preferred embodiments. Prior to or concurrent with genotyping,the genomic sample may be amplified by a variety of mechanisms, some ofwhich may employ PCR. See, e.g., PCR Technology: Principles andApplications for DNA Amplification (Ed. H. A. Erlich, Freeman Press, NY,N.Y., 1992); PCR Protocols: A Guide to Methods and Applications (Eds.Innis, et al., Academic Press, San Diego, Calif., 1990); Mattila et al.,Nucleic Acids Res. 19, 4967 (1991); Eckert et al., PCR Methods andApplications 1, 17 (1991); PCR (Eds. McPherson et al., IRL Press,Oxford); and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159 4,965,188,and 5,333,675, and each of which is incorporated herein by reference intheir entireties for all purposes. The sample may be amplified on thearray. See, for example, U.S. Pat. No. 6,300,070 and U.S. patentapplication Ser. No. 09/513,300, which are incorporated herein byreference.

Other suitable amplification methods include the ligase chain reaction(LCR) (for example, Wu and Wallace, Genomics 4, 560 (1989), Landegren etal., Science 241, 1077 (1988) and Barringer et al. Gene 89:117 (1990)),transcription amplification (Kwoh et al., Proc. Natl. Acad. Sci. USA 86,1173 (1989) and WO88/10315), self-sustained sequence replication(Guatelli et al., Proc. Nat. Acad. Sci. USA, 87, 1874 (1990) andWO90/06995), selective amplification of target polynucleotide sequences(U.S. Pat. No. 6,410,276), consensus sequence primed polymerase chainreaction (CP-PCR) (U.S. Pat. No. 4,437,975), arbitrarily primedpolymerase chain reaction (AP-PCR) (U.S. Pat. Nos. 5,413,909, 5,861,245)and nucleic acid based sequence amplification (NASBA). (See, U.S. Pat.Nos. 5,409,818, 5,554,517, and 6,063,603, each of which is incorporatedherein by reference). Other amplification methods that may be usedinclude: Qbeta Replicase, described in PCT Patent Application No.PCT/US87/00880, isothermal amplification methods such as SDA, describedin Walker et al. 1992, Nucleic Acids Res. 20(7):1691-6, 1992, androlling circle amplification, described in U.S. Pat. No. 5,648,245.Other amplification methods that may be used are described in, U.S. Pat.Nos. 5,242,794, 5,494,810, 4,988,617 and in U.S. Ser. No. 09/854,317 andUS Pub. No. 20030143599, each of which is incorporated herein byreference. In some embodiments DNA is amplified by multiplexlocus-specific PCR. In a preferred embodiment the DNA is amplified usingadaptor-ligation and single primer PCR. Other available methods ofamplification, such as balanced PCR (Makrigiorgos, et al. (2002), NatBiotechnol, Vol. 20, pp. 936-9), may also be used.

Additional methods of sample preparation and techniques for reducing thecomplexity of a nucleic sample are described in Dong et al., GenomeResearch 11, 1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592 andU.S. Patent application Nos. 09/916,135, 09/920,491, 09/910,292, and10/013,598.

Methods for conducting polynucleotide hybridization assays have beenwell developed in the art. Hybridization assay procedures and conditionswill vary depending on the application and are selected in accordancewith the general binding methods known including those referred to in:Maniatis et al. Molecular Cloning: A Laboratory Manual (2^(nd) Ed. ColdSpring Harbor, N.Y., 1989); Berger and Kimmel Methods in Enzymology,Vol. 152, Guide to Molecular Cloning Techniques (Academic Press, Inc.,San Diego, Calif., 1987); Young and Davism, P.N.A.S, 80: 1194 (1983).Methods and apparatus for carrying out repeated and controlledhybridization reactions have been described in U.S. Pat. Nos. 5,871,928,5,874,219, 6,045,996 and 6,386,749, 6,391,623 each of which areincorporated herein by reference

The present invention also contemplates signal detection ofhybridization between ligands in certain preferred embodiments. See U.S.Pat. Nos. 5,143,854, 5,578,832; 5,631,734; 5,834,758; 5,936,324;5,981,956; 6,025,601; 6,141,096; 6,185,030; 6,201,639; 6,218,803; and6,225,625, in U.S. Patent application 60/364,731 and in PCT ApplicationPCT/US99/06097 (published as WO99/47964), each of which also is herebyincorporated by reference in its entirety for all purposes.

Methods and apparatus for signal detection and processing of intensitydata are disclosed in, for example, U.S. Pat. Nos. 5,143,854, 5,547,839,5,578,832, 5,631,734, 5,800,992, 5,834,758; 5,856,092, 5,902,723,5,936,324, 5,981,956, 6,025,601, 6,090,555, 6,141,096, 6,185,030,6,201,639; 6,218,803; and 6,225,625, in U.S. Patent application60/364,731 and in PCT Application PCT/US99/06097 (published asWO99/47964), each of which also is hereby incorporated by reference inits entirety for all purposes.

The practice of the present invention may also employ conventionalbiology methods, software and systems. Computer software products of theinvention typically include computer readable medium havingcomputer-executable instructions for performing the logic steps of themethod of the invention. Suitable computer readable medium includefloppy disk, CD-ROM/DVD/DVD-ROM, hard-disk drive, flash memory, ROM/RAM,magnetic tapes and etc. The computer executable instructions may bewritten in a suitable computer language or combination of severallanguages. Basic computational biology methods are described in, e.g.Setubal and Meidanis et al., Introduction to Computational BiologyMethods (PWS Publishing Company, Boston, 1997); Salzberg, Searles,Kasif, (Ed.), Computational Methods in Molecular Biology, (Elsevier,Amsterdam, 1998); Rashidi and Buehler, Bioinformatics Basics:Application in Biological Science and Medicine (CRC Press, London, 2000)and Ouelette and Bzevanis Bioinformatics: A Practical Guide for Analysisof Gene and Proteins (Wiley & Sons, Inc., 2^(nd) ed., 2001).

The present invention may also make use of various computer programproducts and software for a variety of purposes, such as probe design,management of data, analysis, and instrument operation. See, U.S. Pat.Nos. 5,593,839, 5,795,716, 5,733,729, 5,974,164, 6,066,454, 6,090,555,6,185,561, 6,188,783, 6,223,127, 6,229,911 and 6,308,170. Computermethods related to genotyping using high density microarray analysis mayalso be used in the present methods, see, for example, US Patent Pub.Nos. 20050250151, 20050244883, 20050108197, 20050079536 and 20050042654.

Additionally, the present invention may have preferred embodiments thatinclude methods for providing genetic information over networks such asthe Internet as shown in U.S. patent application Ser. Nos. 10/063,559,60/349,546, 60/376,003, 60/394,574, 60/403,381.

(B) Definitions

Nucleic acids according to the present invention may include any polymeror oligomer of pyrimidine and purine bases, preferably cytosine,thymine, and uracil, and adenine and guanine, respectively. (See AlbertL. Lehninger, Principles of Biochemistry, at 793-800 (Worth Pub. 1982)which is herein incorporated in its entirety for all purposes). Indeed,the present invention contemplates any deoxyribonucleotide,ribonucleotide or peptide nucleic acid component, and any chemicalvariants thereof, such as methylated, hydroxymethylated or glucosylatedforms of these bases, and the like. The polymers or oligomers may beheterogeneous or homogeneous in composition, and may be isolated fromnaturally occurring sources or may be artificially or syntheticallyproduced. In addition, the nucleic acids may be DNA or RNA, or a mixturethereof, and may exist permanently or transitionally in single-strandedor double-stranded form, including homoduplex, heteroduplex, and hybridstates.

“Genome” designates or denotes the complete, single-copy set of geneticinstructions for an organism as coded into the DNA of the organism. Agenome may be multi-chromosomal such that the DNA is cellularlydistributed among a plurality of individual chromosomes. For example, inhuman there are 22 pairs of chromosomes plus a gender associated XX orXY pair.

The term “chromosome” refers to the heredity-bearing gene carrier of aliving cell which is derived from chromatin and which comprises DNA andprotein components (especially histones). The conventionalinternationally recognized individual human genome chromosome numberingsystem is employed herein. The size of an individual chromosome can varyfrom one type to another with a given multi-chromosomal genome and fromone genome to another. In the case of the human genome, the entire DNAmass of a given chromosome is usually greater than about 100,000,000 bp.For example, the size of the entire human genome is about 3×10⁹ bp. Thelargest chromosome, chromosome no. 1, contains about 2.4×10⁸ bp whilethe smallest chromosome, chromosome no. 22, contains about 5.3×10⁷ bp.

A “chromosomal region” is a portion of a chromosome. The actual physicalsize or extent of any individual chromosomal region can vary greatly.The term “region” is not necessarily definitive of a particular one ormore genes because a region need not take into specific account theparticular coding segments (exons) of an individual gene.

An “array” comprises a support, preferably solid, with nucleic acidprobes attached to the support. Preferred arrays typically comprise aplurality of different nucleic acid probes that are coupled to a surfaceof a substrate in different, known locations. These arrays, alsodescribed as “microarrays” or colloquially “chips” have been generallydescribed in the art, for example, U.S. Pat. Nos. 5,143,854, 5,445,934,5,744,305, 5,677,195, 5,800,992, 6,040,193, 5,424,186 and Fodor et al.,Science, 251:767-777 (1991). Each of which is incorporated by referencein its entirety for all purposes.

Arrays may generally be produced using a variety of techniques, such asmechanical synthesis methods or light directed synthesis methods thatincorporate a combination of photolithographic methods and solid phasesynthesis methods. Techniques for the synthesis of these arrays usingmechanical synthesis methods are described in, e.g., U.S. Pat. Nos.5,384,261, and 6,040,193, which are incorporated herein by reference intheir entirety for all purposes. Although a planar array surface ispreferred, the array may be fabricated on a surface of virtually anyshape or even a multiplicity of surfaces. Arrays may be nucleic acids onbeads, gels, polymeric surfaces, fibers such as optical fibers, glass orany other appropriate substrate. (See U.S. Pat. Nos. 5,770,358,5,789,162, 5,708,153, 6,040,193 and 5,800,992, which are herebyincorporated by reference in their entirety for all purposes.)

Preferred arrays are commercially available from Affymetrix under thebrand name GENECHIP® and are directed to a variety of purposes,including genotyping and gene expression monitoring for a variety ofeukaryotic and prokaryotic species. (See Affymetrix Inc., Santa Claraand their website at affymetrix.com.) Methods for preparing sample forhybridization to an array and conditions for hybridization are disclosedin the manuals provided with the arrays, for example, for expressionarrays the GENECHIP Expression Analysis Technical Manual (PN 701021 Rev.5) provides detailed instructions for 3′ based assays and the GeneChip®Whole Transcript (WT) Sense Target Labeling Assay Manual (PN 701880 Rev.2) provides whole transcript based assays. The GeneChip Mapping 100KAssay Manual (PN 701694 Rev. 3) provides detailed instructions forsample preparation, hybridization and analysis using genotyping arrays.In preferred aspects the arrays may be the Mapping 10K, Mapping 100K orMapping 500K arrays or array sets.

In another aspect arrays that may be used in connection with the methodsinclude bead arrays such as those described in Gunderson et al., GenomeRes. 14:870-877 (2004). The methods may be applied to a variety ofgenotyping methods, including, for example, those described in Fan etal., Nat. Rev. Genet. 7:632 (2006) and Fan et al. Methods Enzymol.410:57-73 (2006).

In another aspect the methods may be applied to genotyping assays thatuse a universal array for detection. Such methods include, for example,the Molecular Inversion Probe (MIP) assay and the GoldenGate assay. See,Hardenbol et al., Nature Biotech. 21:673-678 (2003) and Fan et al., ColdSpring Harb. Symp. Quant. Biol. 68:69-78 (2003).

An allele refers to one specific form of a genetic sequence (such as agene) within a cell, a sample, an individual or within a population, thespecific form differing from other forms of the same gene in thesequence of at least one, and frequently more than one, variant siteswithin the sequence of the gene. The sequences at these variant sitesthat differ between different alleles are termed “variants”,“polymorphisms”, or “mutations”. In general, polymorphism is used torefer to variants that have a frequency of at least 1% in a population,while the term mutation is generally used for variants that occur at afrequency of less than 1% in a population. In diploid organisms such ashumans, at each autosomal specific chromosomal location or “locus” anindividual possesses two alleles, a first inherited from one parent anda second inherited from the other parent, for example one from themother and one from the father. An individual is “heterozygous” at alocus if it has two different alleles at the locus. An individual is“homozygous” at a locus if it has two identical alleles at that locus.

Preferred markers have at least two alleles, each occurring at frequencyof preferably greater than 1%, and more preferably greater than 10% or20% of a selected population. A polymorphism may comprise one or morebase changes, an insertion, a repeat, or a deletion. A polymorphic locusmay be as small as one base pair. Polymorphic markers includerestriction fragment length polymorphisms, variable number of tandemrepeats (VNTR's), hypervariable regions, minisatellites, dinucleotiderepeats, trinucleotide repeats, tetranucleotide repeats, simple sequencerepeats, and insertion elements such as Alu. The first identifiedallelic form is arbitrarily designated as the reference form and otherallelic forms are designated as alternative or variant alleles. Theallelic form occurring most frequently in a selected population issometimes referred to as the wildtype form. The most frequent allele mayalso be referred the major allele and the less frequent allele as theminor allele. Diploid organisms may be homozygous or heterozygous forallelic forms. A diallelic polymorphism has two forms. A triallelicpolymorphism has three forms. A polymorphism between two nucleic acidscan occur naturally, or be caused by exposure to or contact withchemicals, enzymes, or other agents, or exposure to agents that causedamage to nucleic acids, for example, ultraviolet radiation, mutagens orcarcinogens. Single nucleotide polymorphisms (SNPs) are positions atwhich two alternative bases occur at appreciable frequency (>1%) in thehuman population, and are the most common type of human geneticvariation.

The term genotyping refers to the determination of the geneticinformation an individual carries at one or more positions in thegenome. For example, genotyping may comprise the determination of whichallele or alleles an individual carries for a single SNP or thedetermination of which allele or alleles an individual carries for aplurality of SNPs. For example, a particular nucleotide in a genome maybe a T in some individuals and a C in other individuals. Thoseindividuals who have a T at the position have the T allele and those whohave a C have the C allele. In a diploid organism the individual willhave two copies of the sequence containing the polymorphic position sothe individual may have a T allele and a C allele or alternatively twocopies of the T allele or two copies of the C allele. Those individualswho have two copies of the C allele are homozygous for the C allele,those individuals who have two copies of the T allele are homozygous forthe T allele, and those individuals who have one copy of each allele areheterozygous. The alleles are often referred to as the A allele, oftenthe major allele, and the B allele, often the minor allele. Thegenotypes may be AA (homozygous A), BB (homozygous B) or AB(heterozygous). Genotyping methods generally provide for identificationof the sample as AA, BB or AB.

Linkage disequilibrium or allelic association means the preferentialassociation of a particular allele or genetic marker with a specificallele, or genetic marker at a nearby chromosomal location morefrequently than expected by chance for any particular allele frequencyin the population. For example, if locus X has alleles A and B, whichoccur at equal frequency, and linked locus Y has alleles C and D, whichoccur at equal frequency, one would expect the combination AC to occurat a frequency of 0.25. If AC occurs more frequently, then alleles A andC are in linkage disequilibrium. Linkage disequilibrium (or LD) mayresult, for example, because the regions are physically close, fromnatural selection of certain combination of alleles or because an allelehas been introduced into a population too recently to have reachedequilibrium with linked alleles.

A marker in linkage disequilibrium can be particularly useful indetecting susceptibility to disease (or other phenotype) notwithstandingthat the marker does not cause the disease. For example, a marker (X)that is not itself a causative element of a disease, but which is inlinkage disequilibrium with a gene (including regulatory sequences) (Y)that is a causative element of a phenotype, can be detected to indicatesusceptibility to the disease in circumstances in which the gene Y maynot have been identified or may not be readily detectable. Studies usingpanels of human SNPs to identify evidence for linkage between genomicregions and disease phenotypes have been described. See, for example,Boyles et al., Am J Med Genet A. 140(24):2776-85 (2006), Klein et al.Science 308: 385 (2005), Papassotiropoulos et al., Science 314:475-478(2006), Craig and Stephan, Expert Rev Mol Diagn 5(2):159-70 (2005) andPuffenberger et al., PNAS 101:11689-94 (2004).

Normal cells that are heterozygous at one or more loci may give rise totumor cells that are homozygous at those loci. This loss ofheterozygosity may result from structural deletion of normal genes orloss of the chromosome carrying the normal gene, mitotic recombinationbetween normal and mutant genes, followed by formation of daughter cellshomozygous for deleted or inactivated (mutant) genes; or loss of thechromosome with the normal gene and duplication of the chromosome withthe deleted or inactivated (mutant) gene.

A homozygous deletion is a deletion of both copies of a gene or of agenomic region. Diploid organisms generally have two copies of eachautosomal chromosome and therefore have two copies of any selectedgenomic region. If both copies of a genomic region are absent the cellor sample has a homozygous deletion of that region. Similarly, ahemizygous deletion is a deletion of one copy of a gene or of a genomicregion.

Genetic rearrangement occurs when errors occur in DNA replication andcross over occurs between nonhomologous regions resulting in geneticmaterial moving from one chromosomal location to another. Rearrangementmay result in altered expression of the genes near the rearrangement.

An aneuploid is a cell whose chromosomal constitution has changed fromthe true diploid, for example, extra copies of a chromosome orchromosomal region.

An individual is not limited to a human being, but may also includeother organisms including but not limited to mammals, plants, bacteriaor cells derived from any of the above.

In some aspects information about one SNP may be used to extrapolate toinformation about another SNP that is part of a haplotype. Informationabout haplotypes, uses of haplotypes and methods of haplotype analysisare described, for example, in de Bakker et al., Nature Genet.37:1217-1223 (2005), Goldstein and Cavalleri, Nature 437:1241-1242(2005), Wang et al., Nat. Rev. Genet. 6:109-118 (2005), Carlson et al.,Nature 429:446-452 (2004) and Clayton et al., Nature Genet. 37:1243-46(2005).

The Whole Genome Sampling Assay (WGSA) reduces the complexity of anucleic acid sample by amplifying a subset of the fragments in thesample. A nucleic acid sample is fragmented with one or more restrictionenzymes and an adapter is ligated to both ends of the fragments. Aprimer that is complementary to the adapter sequence is used to amplifythe fragments using PCR. During PCR fragments of a selected size rangeare selectively amplified. The size range may be, for example, 400-800or 400 to 2000 base pairs. Fragments that are outside the selected sizerange are not efficiently amplified.

The fragments that are amplified by WGSA may be predicted by in silicodigestion and an array may be designed to genotype SNPs that arepredicted to be amplified. Genotyping may be done by allele specifichybridization with probes that are perfectly complementary to individualalleles of a SNP. A set of probes that are complementary to the regionsurrounding each SNP may be present on the array. Perfect match probesare complementary to the target over the entire length of the probe.Mismatch probes are identical to PM probes except for a single mismatchbase. The mismatch position is typically the central position so for a25 base probe the mismatch is position 13.

The methods may be combined with other methods of genome analysis andcomplexity reduction. Other methods of complexity reduction include, forexample, AFLP, see U.S. Pat. No. 6,045,994, which is incorporated hereinby reference, and arbitrarily primed-PCR (AP-PCR) see McClelland andWelsh, in PCR Primer: A laboratory Manual, (1995) eds. C. Dieffenbachand G. Dveksler, Cold Spring Harbor Lab Press, for example, at p 203,which is incorporated herein by reference in its entirety. Additionalmethods of sample preparation and techniques for reducing the complexityof a nucleic sample are described in Dong et al., Genome Research 11,1418 (2001), in U.S. Pat. Nos. 6,361,947, 6,391,592, 6,458,530 and U.S.Patent application Nos. 20030039069, 09/916,135, 09/920,491, 09/910,292and 10/264,945, which are incorporated herein by reference in theirentireties.

The design and use of allele-specific probes for analyzing polymorphismsis described by e.g., Saiki et al., Nature 324, 163-166 (1986);Dattagupta, EP 235,726, Saiki, and WO 89/11548. Allele-specific probescan be designed that hybridize to a segment of target DNA from oneindividual but do not hybridize to the corresponding segment fromanother individual due to the presence of different polymorphic forms inthe respective segments from the two individuals. Hybridizationconditions should be sufficiently stringent that there is a significantdifference in hybridization intensity between alleles, and preferably anessentially binary response, whereby a probe hybridizes to only one ofthe alleles.

(C) Methods for Genotyping Heterozygous Loci

For biallelic polymorphisms in diploid organisms (with alleles A or B)there are generally three available genotype calls, AA, BB or AB. The ABcall, or the heterozygous call, is often the most difficult to make andoften results in a “no call” being made. A “no call” result is often theresult of the genotyping software making a call that is below a userdefined threshold of reliability. Methods are disclosed herein forimproved methods for making heterozygous genotyping calls from intensitydata obtained from probe arrays. In general, the methods involve anormalization step for the intensity measurement obtained for one of thetwo alleles, using an adjustment factor calculated from intensitiesobserved in a training set of samples of known genotype. Often there isan asymmetry observed between the intensity measurement obtained for thetwo alleles and adjusting for the asymmetry can improve the ability tocall heterozygotes. The asymmetry may be the result, for example, ofdifferences in hybridization kinetics between the target and the allelespecific probes for each of the different alleles. In some aspects theprobes may vary only at the interrogation position (the basecomplementary to the polymorphic position) but in others they may haveadditional differences. In assays based on enzymatic discrimination, thedifferences may be the result of differences in enzyme kinetics fordifferent bases. In some aspects the scale of asymmetry is estimated andused to adjust the intensity measurement values for one of the allelesto normalize for the asymmetry.

In a preferred aspect, the method takes into consideration theobservation that the intensity measurement for the A allele and theintensity measurement for the B allele may vary significantly despiteapproximately equal amounts of the two alleles in the starting material.For example, the A allele may have an intensity measurement of about10,000 and the B allele may have an intensity measurement of about6,000. This may result in a no-call because the variation is greaterthan expected. In the methods disclosed herein a training set of samplesof known genotype are used to derive a normalization factor for thisSNP. For example, the training set of individuals of known genotype atthe SNP are interrogated to obtain intensity measurements for the A andB alleles and the measurements are averaged in an allele specific mannerto obtain and average intensity for the A allele and an averageintensity for the B allele, which may be, for example, 10,000 and 5,000.A ratio of the A average to the B average may be calculated(10,000/5,000=2 in this example) and the measurement for B in theunknown sample may be normalized using this number (6,000×2=12,000) andthe normalized measurement used to make the genotyping call. In thisexample, the adjusted measurement for B is 12,000 and the unadjustedmeasurement for A is 10,000 and because the numbers fit the algorithmmodel for a heterozygout, a heterozygous call is made.

Methods for calling the genotype of a biological sequence using dataobtained from a microarray are disclosed herein. The methods are relatedto those disclosed in US Patent Publication Nos. 20050287575 and20050123971, which are incorporated herein by reference in theirentireties.

One of the challenges of genotyping diploid organisms is thatheterozygous loci (AB) are often more difficult to accurately genotypethan homozygous loci (AA or BB). Differences in hybridization behaviorof the allele specific probes for the different alleles may account forsome of this difficulty. Methods for making genotype calls may be biasedagainst making calls for heterozygous loci and may generate “no calls”frequently because the algorithms assume that the intensities or signalfrom the A allele probes and the intensities or signal from the B alleleprobes should be similar and share the same distribution. The methodsdisclosed employ an intensity transformation procedure performed priorto the Dynamic Modeling (DM) algorithm to address asymmetry between theA and B allele probes.

In one embodiment the distribution of PMa/PMb for the reference quartetsis compared to the PMa/PMb for the experimental sample and a P value isobtained for each possible genotype. If the P value for the AB call isat least 0.4 the AB call is supported and the PMb intensity value isadjusted. The difference between the P value for AB should vary from thenext highest P value by at least 0.2. For example, if the P value forthe AB call is 0.8, for AA is 0.0035 and for BB is 0.015 the AB call issupported.

For the training set, the algorithm employs a quartet selection step andan intensity-ratio summarization step. In the quartet selection step, afixed number of quartets are selected based on their quartet levelconcordance derived by comparison with the reference genotype. Thequartet level genotype is calculated using the DM algorithm. In theratio summarization step, PMa/PMb intensity ratio distribution issummarized for AA, AB and BB sub-groups. This summarization requiresthat there are at least two samples for each genotype at that particularSNP. For SNPs that do not meet this criterion, only the quartetselection step is carried out, but no intensity adjustment step on thetest set thereafter.

For the test set, the algorithm consists of a feature extraction step,an evaluation step of whether a selected quartet supports a heterozygouscall, and an intensity adjustment step. In the feature extraction step,only the intensity measures from the k selected quartets (determined bythe training set) is obtained and further used. In the evaluation step,the PMa/PMb ratios from the selected quartets are compared against thecorresponding distribution derived from the training set. Only thosethat show a strong possibility of supporting a heterozygous call arefurther selected for intensity adjustment. Intensity adjustment is doneby multiply the mean and standard deviation of the PMb feature intensityby the mean PMa/PMb ratio of the reference sub-group that calls “AB” onthat particular SNP. In one embodiment, the quartets are selected assupporting a heterozygous call if the PMa/PMb ratio from the test sampleis compared with the reference distribution from AA, AB and BB groupsand the p-value from the comparison with the AB group is the largest andexceeds 0.4 and the difference between this p-value and the secondlargest p-value is greater than 0.2.

After the quartet selection and intensity adjustment, DM is applied onthis transformed intensity set and genotypes and significance arerecalculated. In the experiments using whole genome target, we observesubstantial improvement in the genotyping quality after this intensityadjustment. The improvement is especially significant on heterozygouscalls.

FIG. 1 shows a schematic of the method. In step [101] a genotype isderived from a training set using the genotyping algorithm. The genotypefrom [101] is compared with the reference genotype to obtain featurelevel concordance for the training set [103]. The best features areselected from the training set based on concordance [105]. The (Aintensity)/(B intensity) distribution from AA, AB and BB sub-groups inthe training set are summarized [107]. Intensities are extracted fromthe selected K best features for the test set [109]. The (Aintensity)/(B intensity) for the test set is compared with the referencefrom the training set to decide the likelihood of supporting AB call forthe test set [111]. If AB is supported the B allele intensity isadjusted by the ratio from the reference AB group [115]. If AB is notsupported the original intensity for both A and B is maintained [117].The intensity values are fed back into a genotyping algorithm to getgenotype and significance [121]. Steps 101, 103, 105 and 107 areperformed on the training set. Steps 109, 111, 113, 115, 117 and 121 areperformed on the test set. The genotypes of the training set are known.The genotypes of the test set are unknown.

FIG. 2 shows a schematic of an embodiment of the method as applied to agenotyping array using allele specific probes. The intensities arehybridization intensities that measure the amount of a labeled targetthat is hybridized to individual probe features. In step [201] a quartetlevel genotype is derived from a training set using the DM algorithm.The genotype from [201] is compared with the reference genotype toobtain a quartet level concordance for the training set [203]. The bestquartets are selected from the training set based on concordance [205].The PMa/PMb distribution from AA, AB and BB sub-groups in the trainingset are summarized [207]. Intensities are extracted from the selected Kbest quartets for the test set [209]. PMa/PMb for the training set iscompared with the reference from the training set to decide thelikelihood of supporting AB call for the test set [211]. If AB issupported the PMb intensity is adjusted by the ratio from the referenceAB group [215]. If AB is not supported the original intensity ismaintained [217]. The intensity values are feed back into DM to getgenotype and significance [221]. Steps 101, 103, 105 and 107 areperformed on the training set. Steps 209, 211, 213, 215, 217 and 221 areperformed on the test set. The genotypes of the training set are known.The genotypes of the test set are unknown.

FIG. 2. Each individual in the training set is evaluated at the quartetlevel to get a predicted genotype for each SNP for each individual. Thegenotypes are compared to the reference genotype to obtain a concordancemeasure for each SNP in the training set. For a given SNP the K quartetswith the highest concordance are selected for further analysis. In apreferred aspect K is a constant and may be, for example, 3, 4, 5, 6, or7. K is less than the total number of quartets and represents a highperforming subset of quartets for a given SNP. In another embodiment Kmay vary from SNP to SNP and may be determined by a thresholdconcordance, for example, all quartets that have a concordance betweenthe reference genotype and the predicted quartet level genotype withinthe reference set, may be selected. Summarize the distribution ofPMa/PMb for the selected quartets for each SNP and each of the threegenotypes, AA, AB and BB for the reference set. So for each SNP you havea selected set of the K best quartets and for each quartet a mean andstandard deviation for each of the three genotypes based on thereference set of samples. For the unknown sample intensity informationis extracted from the K best quartets for each SNP. PMa/PMb iscalculated for each quartet and compared to the PMa/PMb values obtainedfor that quartet in the reference set for AA, AB or BB referencegenotypes. The experimental PMa/PMb value is compared to the PMa/PMbvalue for the AB, AA and BB genotypes to determine if an AB call issupported. This is done at the quartet level and the reference genotypelevel. The AB call is supported if the ratio from the experimentalsample is closer to the AB value than to the AA or BB value by at leasta minimum threshold. If the AB call is supported, the PMb intensity isadjusted by multiplying the PMb intensity value by the PMa/PMb valuefrom the AB reference set for the genotype. If the AB call is notsupported no adjustment to the PMb value is made. After the adjustmentstep the intensities are used to make genotyping calls using the DMalgorithm (Di et al., Bioinformatics 21:1958-1963 (2005). Otheralgorithms may be used, for example, Hua et al., bioinformatics 2006(PMID 17062589) discloses an expectation-maximization algorithm for usewith high density SNP arrays, Rabbee and Speed Bioinformatics 22:7-12(2006) discloses the RLMM algorithm as applied to SNP arrays, this is arobustly fitted, linear model that uses the Mahalanobis distance forclassification. In another embodiment the algorithm is based on BRLMM asdescribed in BRLMM: an Improved Genotype Calling Method for the GeneChipHuman Mapping 500K Array set, white paper available at Affymetrix website, revision date 2006-04-14, see also, U.S. Provisional Patentapplication No. 60/744,002 filed Mar. 30, 2006.

Related methods of analysis of genotype information are also disclosedin U.S. Patent Publication Nos. 20050287575, 20050250151, 20050227244,20050222777, 20050208555, and 20050164270.

Also, each embodiment of probe array may include a plurality of probesets each comprising a plurality of probes enabled to interrogate thenucleotide composition of each SNP position. Also, some embodimentsinclude one or more probe sets enabled to interrogate sequencecomposition associated with a complementary sequence (i.e. complementarysequence by Watson-Crick base paring rules) region on each of the twostrands of DNA, for example, the sense strand and the anti-sense strandof DNA.

In another aspect, the methods may be applied to a genotyping systemthat uses single base extension (SBE) or allele specific primerextension (ASPE). SBE has been described, for example, in Fan et al.,Genome Res. 10:853-60 (2000). For SBE, a single locus specific primerthat hybridizes immediately adjacent to the polymorphism is extendedwith a base that is complementary to the polymorphic base. The base thatis added is identified to identify which allele of the polymorphism ispresent. For ASPE, each allele of the polymorphisms is targeted by adifferent allele specific probe. The probe hybridizes to the region ofthe target including the polymorphism and is extended to incorporatelabeled nucleotides. ASPE has been described, for example, in Patinen etal., Genome Res. 10:1031 (2000). ASPE methods for genotyping usingarrays of probes attached to beads have been described, for example, inGunderson et al Nat. Genet. 37, 549-554 (2005). For a description ofother SNP genotyping methods to which the disclosed methods may beapplied see Syvanen, Nat. Genet. Suppl: S5-10 (2005).

In one aspect each SNP is interrogated by a probe set that includes oneor more probe quartets. Each probe quartet is comprised of a PerfectMatch (PM) and a Mismatch (MM) probe for each allele. The probes of aquartet are complementary to the same strand (sense or antisense) butdifferent quartets may be complementary to different strands. A probeset may include one or more quartets to the sense strand and one or morequartets to the antisense strand. In a preferred aspect the probe setfor each SNP includes 7 to 10 probe quartets. Preferably theoligonucleotides of a quartet are 20 to 50 bases, more preferably 20-30bases and most preferably 25 bases in length. Each probe is located in adifferent feature of the array and there are multiple copies of theprobe in the feature, preferably more than 1 million copies of the probein that probe's feature. The array may have more than 1 million andpreferably more than 2.5 million features. In preferred aspects thearray contains probe sets for more than 50,000 different SNPs. Exemplaryarrays include the Affymetrix Mapping 100K array set. See Data SheetGeneChip® Human Mapping 100K Set available from Affymetrix (PN 701674Rev. 3) and the Mapping 100K Assay Manual.

In a preferred aspect, genomic material is hybridized to arrays withoutreduction of complexity. These whole genome hybridization experimentsmake genotype calling more difficult. In one example, differentstringencies of washes were used. For the test set the wash was in0.6×SSPE (90 mM NaCl, 6 mM NaH2Po4) and the training set was washed in50 mM Tris pH 8.3 with varying salt concentrations of either 75 mM, 100mM, or 125 mM NaCl. The samples tested were 16 HapMap samples and 150 μgof whole genome target was hybridized to the arrays. Hybridization wasat 50° C. for 18 hours in 0.6×SSPE. The results showed that there was anincreased asymmetry between PMa and PMb intensity and adjusting tominimize the asymmetry improved heterozygous call efficiency.

CONCLUSION

Methods of determining genotypes of polymorphisms are disclosed. Allcited patents, patent publications and references are incorporatedherein by reference for all purposes.

It is to be understood that the above description is intended to beillustrative and not restrictive. Many variations of the invention willbe apparent to those of skill in the art upon reviewing the abovedescription. By way of example, the invention has been describedprimarily with reference to the use of a high density oligonucleotidearray, but it will be readily recognized by those of skill in the artthat other nucleic acid arrays, other methods of measuring signalintensity resulting from genomic DNA could be used. The scope of theinvention should, therefore, be determined not with reference to theabove description, but should instead be determined with reference tothe appended claims, along with the full scope of equivalents to whichsuch claims are entitled.

1) A method for calling the genotype of a sample at a selectedpolymorphism in a sample using a genotyping array, comprising: (a)obtaining intensity measurements for allele A and for allele B for aplurality of polymorphisms in a plurality of training samples, whereinthe genotype of each polymorphism in the plurality in each trainingsample is known of known genotype, wherein the intensity measurementsrepresent intensity of signal associated with one or more features onsaid genotyping array; (b) making a genotype call for each of a saidpolymorphisms in each of the training samples using the intensitymeasurements for allele A and for allele B obtained in (a); (c)comparing the genotype call with the known genotype to identifyindividuals where the correct genotype call was made; (d) using theintensity measurements from the individuals identified in (c) tocalculate a ratio of intensity measurement for allele A to intensitymeasurement for allele B, for the training samples for each sub-group ofAA, AB and BB to obtain an AA reference ratio, an AB reference ratio anda BB reference ratio for each of said polymorphisms; (e) hybridizing atest sample to the genotyping array to obtain hybridization intensityvalues for the A allele and for the B allele for each of saidpolymorphisms in the test sample; (f) calculate a ratio of the intensitymeasurement for the A allele to the B allele for each of saidpolymorphisms in the test sample and compare the ratio to the referenceratios for AA, AB and BB obtained for that polymorphism in (d) todetermine the likelihood that the polymorphism is AB; (g) identify asubset of the polymorphisms in the test sample that are likely to be AB,wherein a polymorphism is identified as being likely to be AB if thelikelihood that the polymorphism is AB is greater than a selectedthreshold; (h) adjust the intensity measurement of the B allele by thereference ratio for the AB group for that polymorphism from the trainingset to obtain an adjusted intensity measurement for the B allele, foreach polymorphism in the subset of polymorphisms identified in (g); and(i) generate a genotype call for each polymorphisms identified in (g)using the use the adjusted intensity measurement for the B allele. 2.The method of claim 1 wherein said polymorphisms are single nucleotidepolymorphisms.
 3. The method of claim 1 wherein said intensitymeasurement is obtained by measuring the amount of a labeled target thatis hybridized to one or more allele specific probes.
 4. The method ofclaim 1 wherein said intensity measurement is obtained by measuring theamount of label incorporated by extending a probe with one or morelabeled nucleotides.
 5. The method of claim 4 wherein the probe isextended enzymatically in a template dependent manner.
 6. A system forcalling the genotype of a sample using the method of claim 1,comprising: a scanner that generates allele specific intensitymeasurements for one or more polymorphisms from an array of probes; anda computer comprising system memory with executable code stored thereon,wherein the executable code is enabled to perform one or more of thesteps of claim
 1. 7. A method for genotyping a polymorphism in a testindividual comprising: (a) obtaining allele specific intensitymeasurements for a first allele of the polymorphism and for a secondallele of the polymorphism in a training set comprising a plurality ofindividuals of known genotype for said polymorphism; (b) grouping theindividuals in the training set into groups according to genotype,wherein each individual is assigned to one of the following groups:homozygous for the first allele, homozygous for the second allele orheterozygous; (c) calculating a summary ratio of the intensitymeasurements for said first allele to the intensity measurements forsaid second allele for each group to obtain a first summary ratio forthe group that are homozygous for said first allele, a second summaryratio for the group that are homozygous for said second allele and athird summary ratio for the group that are heterozygous; (d) obtainingan allele specific intensity for said first allele and for said secondallele in said test individual and calculating a test ratio for saidtest individual; (e) adjusting the allele specific intensity value forsaid first allele according to the third summary ratio obtained in (c)if said test ratio is closer to the third summary ratio than to thefirst or second summary ratios; and (e) making a genotype call for saidpolymorphism using the adjusted value obtained in (e) for said firstallele and the intensity value obtained in (d) for the second allele. 8.The method of claim 7 wherein the genotype call of step (e) is madeusing a dynamic modeling algorithm.
 9. The method of claim 7 wherein thegenotype call of step (e) is made using an expectation-maximizationalgorithm.
 10. A method for calling the genotype of a sample at aselected polymorphism in a sample using a genotyping array, comprising:(a) obtaining hybridization intensity values for said genotyping arrayfor each of a set of training samples comprising a plurality of trainingsamples of known genotype; (b) making a genotype call for each of aplurality of SNPs in the each of the training samples using thehybridization intensity values from individual probe quartets; (c)comparing the genotype call with the known genotype for each probequartet to identify a plurality of K best probe quartets for each SNP,where K is at least 1, wherein probe quartets are selected as best probequartets if the genotype call made using said quartet has highconcordance with the known genotype for that SNP; (d) calculating adistribution of (intensity A)/(intensity B) distribution for thetraining samples for each sub-group of AA, AB and BB to obtain an AAreference distribution, an AB reference distribution and a BB referencedistribution; (e) hybridizing a test sample to the genotyping array toobtain hybridization intensity values for said K best probe quartets;(f) calculate (intensity A)/(intensity B) for each quartet and comparewith the reference distributions for AA, AB and BB to determine thelikelihood that the polymorphism is AB; (g) if the likelihood that thepolymorphism is greater than a selected threshold adjust the intensityof intensity B by the (intensity A)/(intensity B) ratio from the ABgroup from the reference set to obtain an adjusted allele B intensity;and (h) use the adjusted intensity B value to generate a genotype callusing a selected algorithm.
 11. The method of claim 10 wherein K isselected from 2, 3, 4, 5, 6 or
 7. 12. The method of claim 11 wherein Kis the same for each SNP in the plurality.
 13. The method of claim 10wherein K is selected for each SNP and is the number of quartets forthat SNP that predict the correct genotype in the training set with aminimum concordance.
 14. The method of claim 10 wherein the selectedalgorithm is a dynamic modeling algorithm.
 15. The method of claim 10wherein the selected algorithm is an expectation-maximization algorithm.